Cardiac allograft acute cellular rejection (ACR) remains a significant source of morbidity and mortality within the first year after heart transplantation. Afterwards, cardiac allograft vasculopathy (CAV), as a result of chronic vascular rejection, is the major cause of morbidity and mortality. Within the first year post-transplantation, almost two-thirds of recipients will experience at least one rejection episode and approximately one third of these patients will have multiple episodes. At five years post-transplantation, nearly 50% of survivors will have CAV. The clinical symptoms of ACR are relatively nonspecific (fatigue, dyspnea, low grade fever). Most CAV patients remain asymptomatic until they develop serious problems such as myocardial infarction, heart failure, ventricular dysrhythmias or sudden cardiac death. No widely accepted noninvasive method exists for the accurate diagnosis of acute or chronic cardiac rejection. The current gold standard for the diagnosis of ACR remains right ventricular endomyocardial biopsy (EBx), an invasive method of diagnosis subject to morbidity and random sampling and interpretation error. Likewise, the gold standard for diagnosing CAV is cardiac catheterization with intravascular ultrasound, an invasive procedure also subject to morbidity. We are applying functional genomics, detection of cell free donor DNA, and peptidomics to study ACR and CAV. By correlating putative biomarkers with clinical, histological, and imaging based evidence of allograft disease we hope to build a database comprised of genomic and peptidomic data relevant to the immunologic relationship between the donor organ and recipient. Published reports have established that detection of donor DNA in recipient blood can serve as a diagnostic tool of graft injury. The level of donor DNA measured as percentage of circulating cell-free donor DNA (%ccfdDNA) has the potential to accurately diagnose ACR with a high sensitivity and specificity, possibly at times earlier than the diagnosis obtained by conventional EBxs. The ability of cell free DNA to diagnose graft injury early opens a new window to re-examine markers of rejection. These markers are traditionally evaluated using biopsy results, often positive late during rejection. %ccfdDNA offers an opportunity to better characterize our analyses. In collaboration with the NHLBI Laboratory of Transplantation Genomics, we are embarking on biomarker discovery using our collected samples and their recently developed genomic approaches. Peptidomics has also emerged as a viable and promising diagnostic tool and has been applied to diseases such as tuberculosis and chemotherapy-induced heart disease. This tool, has been used to identify peptide biomarkers of diagnostic and prognostic significance in these disease entities. We are planning to collaborate with the Arizona State Universitys Virginia G. Piper Center for Personalized Diagnostics to explore biomarker discovery schemes using our collected samples and their recently developed integrated nanotechnique-based peptidomic strategies. Blood and urine specimens were obtained serially from heart transplant recipients during periods of immunological tolerance of the allograft (no ACR) and immunologic intolerance of the allograft (ACR) and from heart transplant recipients with and without CAV. The samples will be analyzed to determine whether unique gene and/or protein/peptide expression patterns are associated with each state. In the latter phase of the project we hope to translate these profiles into an acceptable test for acute and chronic cardiac allograft rejection. In addition to developing a biomarker approach to the diagnosis of rejection in cardiac transplant patients, expression profiling has the potential to identify immunoregulatory pathways that can serve as new targets for immunosuppressive therapy (rational drug development). On Oct 29, 2015 the protocol was closed to new transplant candidates with a total enrollment of 187 subjects. As of December 15, 2015, we no longer obtained samples with biopsies from those we followed that have been transplanted and no longer followed the subjects who remained on the transplant wait. We continue to collect clinical information (i.e. survival) and also continue to recruit healthy volunteers to match our remaining unmatched patients. Our protocol has created at NIH a bio-bank of samples from transplant subjects with well-characterized clinical phenotypes. The protocol has obtained a minimum of one year of sample collections from all subjects being actively followed that have been transplanted. We have collected blood and urine samples during 621 EBxs. On average from each biopsy time point we stored 5 plasma tubes, 4 serum tubes, 1 tube of RNA, and 1 urine sample. During the 2013-14 reporting period we entered into a collaboration with the Laboratory of Transplantation Genomics (LTG) of the NHLBI. During the 2014-2015 reporting period we embarked on a collaborative biomarker discovery study with the Houston Methodist Hospital Research Institute (THMHRI; Material Transfer Agreement (MTA) established Aug 14, 2015) focusing on using peptidomics and exosomes. The goal of both collaborations is to identify markers of graft injury. Over the reporting period 2015-16 we shared a subset of our plasma (1,205) and urine samples (74) with LTG. The urine samples were analyzed to determine the physical properties of cell-free DNA in urine to aid in protocol development (JHLT 35(4S):S16, 2016). From the plasma samples, 101 have been analyzed to date for cell free DNA (JHLT 35(4S):S161, 2016; JHLT 36(9): 1004-12, 2017). The remainder were awaiting external validation of biopsy specimens to confirm rejection status before proceeding further. These consensus histopathology reads were completed in the 2016-17 reporting period (see below). During the 2016-2017 reporting period, we retrieved histologic slides that were coincident to 350 bio-specimen time-points and performed consensus histopathology reads. Consensus reads were performed by two expert transplant pathologists. The data produced from the consensus reads and the clinical histopathology dataset from Inova Health System enabled concordance analyses. Through these analyses, we are now able to identify bio-sample time-points with concordance biopsy results for the collaborative studies with LTG and potentially with the Biodesign Institute at Arizona State University (The Houston collaborator (see 2014-15 reporting period) has since moved to Arizona State University). Over the 2017 -2018 reporting period, aided by the consensus reads performed previously (2016-17 reporting period), we have identified 3 subjects in our cohort with allograft antibody-mediated rejection (cardiac AMR) and have analyzed their plasma samples for %ddcfDNA. We are in the process of combining this cohort with two other cohorts from Stanford and LTG in anticipation of increasing the sample size for further analysis. In addition, we continue to explore a collaborative study with the Biodesign Institute at Arizona State University (see 2016-17 reporting period). The collaborator recently moved institutions and is in the process of reestablishing his techniques at the new institution. Under a Materials Transfer Agreement we would plan to share a subset (639) of our plasma samples with the Arizona group.